SUMMARY Stroke is the fifth leading cause of death and the leading cause of adult long-term disability in the U.S. and other industrialized nations. Yet only one therapeutic agent (the clot-dissolving drug tPA) is approved for acute treatment. Among the critical injurious factors in stroke, oxidative stress is thought to contribute to the terminal steps of tissue damage. Antioxidants and free radical scavengers are highly protective in animal stroke models. Yet, for poorly understood reasons, they have shown limited or no benefits in clinical trials. In the prior NIH- sponsored project, we collected strong data which may help to revise our understanding of the mechanisms contributing to oxidative injury in ischemia: (1) In a rat model of stroke, we found that the superoxide dismutase (SOD) mimetic tempol was more protective than clinically used antioxidants. (2) Potent protection by tempol correlated with reduction of redox-sensitive glutamate release in the ischemic penumbra. (3) Glutamate release was mediated by at least two redox-sensitive mechanisms: volume-regulated anion channels (VRAC) and Ca2+- dependent changes in membrane permeability. (4) The glutamate-permeable VRAC was composed of proteins from the leucine-rich repeat-containing family 8 (LRRC8). We have assembled a synergistic team of investi- gators and propose to use highly innovative molecular and animal tools to test the HYPOTHESIS that reactive oxygen species (ROS, particularly superoxide anions) propagate and amplify stroke injury via stimulation of redox-sensitive glutamate release in the clinically relevant penumbra. In the planned studies we will address the following critical questions: (1) Is the LRRC8A-containing VRAC a viable target for neuroprotection in stroke? (2) Is glutamate release via the heteromeric LRRC8 channels responsible for tissue injury? (3) What is the chemical nature of the tempol-targeted ROS and the cellular site of their production? (4) Does glutamate release via VRAC drive disruption at the neurovascular interface (changes in blood flow and BBB integrity)? The immediate goal of the proposed work is to identify new molecular mechanisms that govern oxidative brain injury and determine the protective actions of antioxidants. Our long-term objective is to provide a blueprint for the development of new effective stroke therapies based on SOD mimetics and/or VRAC blockers.